Sex hormone-dependent and -independent regulation of serum BAFF and TNF in cohorts of transgender and cisgender men and women.

Background: The risk of inflammatory diseases is sex-dependent, but it remains unknown whether this is due to the impact of sex hormones or sex chromosomes. Transgender individuals represent a unique cohort for studying the relative influence of endocrine and chromosomal factors. Here we compared serum levels of B-cell activating-factor (BAFF) and tumor necrosis factor (TNF) in transgender men (TM), transgender women (TW), cisgender women (CW) and cisgender men (CM). Methods: BAFF and TNF were measured in the serum of 26 CW, 30 CM, 27 TM and 16 TW individuals. To determine the responsiveness of immune cells, TNF was measured in bacterial lipopolysaccharide (LPS)-treated peripheral leukocytes. Results: BAFF was higher in CF (998 pg/mL) and TW (973 pg/mL) compared to CM (551 pg/mL) (P < 0.0001) and TM (726 pg/mL) (P < 0.0001). No difference in BAFF levels was shown between subjects grouped according to the number of X chromosomes. TNF was higher in CM (174 pg/mL) than TW (2.3 pg/mL) (P = 0.027) and TM (27.4 pg/mL) (P = 0.028). LPS-induced TNF was higher in CM (2524 pg/mL) and TM (2078 pg/mL) than in CW (1332 pg/mL) (both P < 0.0001) and TW (1602 pg/mL) (both P = 0.009). Discussion: Sex hormones and sex chromosomes have different impacts on cytokines involved in the sex-dependent inflammatory response. The concentration of BAFF and LPS-stimulated TNF secretion depended on sex hormone levels, whereas basal TNF was regulated by both sex hormone-dependent and -independent factors.

Novel protein markers of androgen activity in humans: proteomic study of plasma from young chemically castrated men.

BACKGROUND: Reliable biomarkers of androgen activity in humans are lacking. The aim of this study was, therefore, to identify new protein markers of biological androgen activity and test their predictive value in relation to low vs normal testosterone values and some androgen deficiency linked pathologies. METHODS: Blood samples from 30 healthy GnRH antagonist treated males were collected at three time points: (1) before GnRH antagonist administration; (2) 3 weeks later, just before testosterone undecanoate injection, and (3) after additional 2 weeks. Subsequently, they were analyzed by mass spectrometry to identify potential protein biomarkers of testosterone activity. Levels of proteins most significantly associated with testosterone fluctuations were further tested in a cohort of 75 hypo- and eugonadal males suffering from infertility. Associations between levels of those markers and cardiometabolic parameters, bone mineral density as well as androgen receptor (AR) CAG repeat lengths, were explored. RESULTS: Using receiver operating characteristic analysis, 4-hydroxyphenylpyruvate dioxygenase (4HPPD), insulin-like growth factor-binding protein 6 (IGFBP6), and fructose-bisphosphate aldolase (ALDOB), as well as a Multi Marker Algorithm, based on levels of 4HPPD and IGFBP6, were shown to be best predictors of low (<8 nmol/l) vs normal (>12 nmol/l) testosterone. They were also more strongly associated with metabolic syndrome and diabetes than testosterone levels. Levels of ALDOB and 4HPPD also showed association with AR CAG repeat lengths. CONCLUSIONS: We identified potential new protein biomarkers of testosterone action. Further investigations to elucidate their clinical potential are warranted. FUNDING: The work was supported by ReproUnion2.0 (grant no. 20201846), which is funded by the Interreg V EU program.

Although it is best known for its role in developing male sex organs and maintaining sexual function, the hormone testosterone is important for many parts of the human body. A deficiency can cause an increased risk of serious conditions such as diabetes, cancer and osteoporosis. Testosterone deficiency can develop due to disease or age-related changes, and men affected by this can be given supplements of this hormone to restore normal levels. The most common way to test for testosterone deficiency is by measuring the concentration of the hormone in the blood. However, this does not accurately reflect the activity of the hormone in the body. This may lead to men who need more testosterone not receiving enough, and to others being unnecessarily treated. Several factors may lead to discrepancy between testosterone concentration in blood and its physiological activity. One of the most common is obesity. Additionally, certain genetic factors, which cannot be controlled for yet, regulate sensitivity to this hormone: some people do well at low levels, while others need high concentrations to be healthy. Therefore, to improve the diagnosis of testosterone deficiency it is necessary to identify biological markers whose levels act as a proxy for testosterone activity. Giwercman, Sahlin et al. studied the levels of a large number of proteins in the blood of 30 young men before and after blocking testosterone production. The analysis found three proteins whose concentrations changed significantly after testosterone deprivation. Giwercman, Sahlin et al. then validated these markers for testosterone deficiency by checking the levels of the three proteins in a separate group of 75 men with fertility problems. The results also showed that the three protein markers were better at predicting diabetes and metabolic syndrome than testosterone levels alone. These newly discovered markers could be used to create a test for measuring testosterone activity. This could help to identify deficiencies and finetune the amount of supplementary hormone given to men as treatment. However, further research is needed to understand the clinical value of such a test in men, as well as women and children.

Short-term effect of pharmacologically induced alterations in testosterone levels on common blood biomarkers in a controlled healthy human model.

Testosterone deficiency in males is associated with serious comorbidities such as cardiovascular disease, diabetes type two, and also an increased risk of premature death. The pathogenetic mechanism behind this association, however, has not yet been clarified and is potentially bidirectional. The aim of this clinical trial was to gain insight into the short-term effect of changes in testosterone on blood analytes in healthy young men. Thirty healthy young male volunteers were recruited and monitored in our designed human model. Blood sampling was performed prior to and 3 weeks after pharmacological castration with a gonadotropin-releasing hormone antagonist. Subsequently, testosterone replacement with 1000 mg testosterone undecanoate was given and additional blood samples were collected 2 weeks later. The alterations in the levels of 37 routine biomarkers were statistically analysed. Eight biomarkers changed significantly in a similar manner as testosterone between the time points (e.g. prostate specific antigen, creatinine and magnesium), whereas seven other markers changed in the inverse manner as testosterone, including sexual hormone-binding globulin, urea, aspartate aminotransferase and alanine aminotransferase. Most of our results were supported by data from other studies. The designed controlled human model yielded changes in known biomarkers suggesting that low testosterone has a negative effect on health in young healthy men.

High prevalence of hypogonadism and associated impaired metabolic and bone mineral status in subfertile men.

OBJECTIVE: It is yet unknown to which degree young subfertile men present with signs of hypogonadism and whether low testosterone concentration, like in older men, is associated with risk of osteoporosis and metabolic derangements in those subjects. The objective was therefore to investigate the prevalence of hypogonadism and its association with metabolic and bone parameters in young subfertile men. DESIGN: Cross-sectional case-control study. PATIENTS: Men from infertile couples (n = 192); 18-50 years; sperm concentration <20 × 10(6) /ml and population-based age-matched controls (n = 199). MEASUREMENTS: Blood sampling, anthropometrics, blood pressure, ankle-brachial index and assessment by dual-energy X-ray absorptiometry were undertaken. Odds ratios of biochemical hypogonadism (total testosterone <8·0 nmol/l and/or luteinizing hormone ≥8·6 IU/l and/or ongoing androgen replacement therapy) were calculated. Serum concentrations of sex hormones, lipids, glucose, insulin and HbA1c were assessed and bone mineral density (BMD) evaluated. RESULTS: Compared to controls, the risk of hypogonadism was increased among subfertile men (OR 10; 95% CI, 5·1, 22), being highest in those with nonobstructive azoospermia. Hypogonadal men had higher HbA1c concentration (mean diff. 2·8 mmol/mol; 95% CI, 0·64, 4·9; P = 0·011) and lower lumbar spine BMD (mean diff. 0·05 g/cm(2) ; 95% CI, 0·01, 0·10; P = 0·032) compared to eugonadal subfertile men, even more pronounced in subfertile men with subnormal testosterone levels. CONCLUSION: Young subfertile men have 10 times increased OR of hypogonadism, which is linked to increased HbA1c and decreased bone mineralization. Endocrine assessment and, if needed, measures to prevent metabolic sequelae and osteoporosis should be included in the routine management of men from infertile couples.

Toll-like receptor 4 promoter polymorphisms: common TLR4 variants may protect against severe urinary tract infection.

BACKGROUND: Polymorphisms affecting Toll-like receptor (TLR) structure appear to be rare, as would be expected due to their essential coordinator role in innate immunity. Here, we assess variation in TLR4 expression, rather than structure, as a mechanism to diversify innate immune responses. METHODOLOGY/PRINCIPAL FINDINGS: We sequenced the TLR4 promoter (4,3 kb) in Swedish blood donors. Since TLR4 plays a vital role in susceptibility to urinary tract infection (UTI), promoter sequences were obtained from children with mild or severe disease. We performed a case-control study of pediatric patients with asymptomatic bacteriuria (ABU) or those prone to recurrent acute pyelonephritis (APN). Promoter activity of the single SNPs or multiple allelic changes corresponding to the genotype patterns (GPs) was tested. We then conducted a replication study in an independent cohort of adult patients with a history of childhood APN. Last, in vivo effects of the different GPs were examined after therapeutic intravesical inoculation of 19 patients with Escherichia coli 83972. We identified in total eight TLR4 promoter sequence variants in the Swedish control population, forming 19 haplotypes and 29 genotype patterns, some with effects on promoter activity. Compared to symptomatic patients and healthy controls, ABU patients had fewer genotype patterns, and their promoter sequence variants reduced TLR4 expression in response to infection. The ABU associated GPs also reduced innate immune responses in patients who were subjected to therapeutic urinary E. coli tract inoculation. CONCLUSIONS: The results suggest that genetic variation in the TLR4 promoter may be an essential, largely overlooked mechanism to influence TLR4 expression and UTI susceptibility.

A genetic basis of susceptibility to acute pyelonephritis.

BACKGROUND: For unknown reasons, urinary tract infections (UTIs) are clustered in certain individuals. Here we propose a novel, genetically determined cause of susceptibility to acute pyelonephritis, which is the most severe form of UTI. The IL-8 receptor, CXCR1, was identified as a candidate gene when mIL-8Rh mutant mice developed acute pyelonephritis (APN) with severe tissue damage. METHODS AND FINDINGS: We have obtained CXCR1 sequences from two, highly selected APN prone patient groups, and detected three unique mutations and two known polymorphisms with a genotype frequency of 23% and 25% compared to 7% in controls (p<0.001 and p<0.0001, respectively). When reflux was excluded, 54% of the patients had CXCR1 sequence variants. The UTI prone children expressed less CXCR1 protein than the pediatric controls (p<0.0001) and two sequence variants were shown to impair transcription. CONCLUSIONS: The results identify a genetic innate immune deficiency, with a strong link to APN and renal scarring.

Reduced toll-like receptor 4 expression in children with asymptomatic bacteriuria.

Toll-like receptor (TLR) 4 is essential for the defense against infection with gram-negative pathogens, but reduced TLR4 expression has not been linked to altered disease susceptibility in humans. In mice, Tlr4 controls the mucosal response to Escherichia coli urinary tract infections. Inactivation of mouse Tlr4 causes an asymptomatic carrier state resembling asymptomatic bacteriuria (ABU). The present study compared neutrophil TLR4 expression levels between children with ABU (n=17) and age-matched control subjects (n=24), and significantly lower levels were detected in the patients with ABU. We also found elevated levels of the TLR4 adaptor protein TRIF and reduced levels of the TLR4-inhibitor SIGIRR in the patients with ABU, but MyD88 and TRAM levels were not significantly altered. Altered TLR4 and adaptor protein expression might impair TLR4 signaling and explain the weak mucosal response to urinary tract infection in patients who develop ABU rather than symptomatic disease.

Inherited susceptibility to acute pyelonephritis: a family study of urinary tract infection.

BACKGROUND: Urinary tract infections (UTIs) are important causes of morbidity and death. The present study investigated whether genetic factors influence susceptibility to acute pyelonephritis (APN). CXCR1 expression was investigated as a factor predisposing to APN, because low CXCR1 expression has been associated with disease susceptibility in mice and disease-prone children. METHODS: The families of APN-prone children (n=130) and of age-matched control subjects without UTI (n=101) were studied. Three-generation pedigrees of UTI-associated morbidity were established by means of structured interviews of the families. CXCR1 expression was quantified by flow cytometric analysis of peripheral blood neutrophils obtained from family members and control subjects. RESULTS: APN was significantly more common in the family members of the APN-prone children (20 [15%] of 130 family members) than in the relatives of the control subjects (3 [3%] of 101 family members) (P<.002). Acute cystitis, in contrast, occurred with equal frequency in both groups (19%; P=1.0). Some families included many affected individuals, consistent with a dominant pattern of inheritance, whereas other families showed a recessive pattern of disease susceptibility. CXCR1 expression was significantly lower in the APN-prone children and in their relatives than in pediatric and adult control subjects (P<.0001). CONCLUSIONS: Our results suggest that susceptibility to APN is inherited and that low CXCR1 expression might predispose to disease.

Do type 1 fimbriae promote inflammation in the human urinary tract?

Type 1 fimbriae have been implicated as virulence factors in animal models of urinary tract infection (UTI), but the function in human disease remains unclear. This study used a human challenge model to examine if type 1 fimbriae trigger inflammation in the urinary tract. The asymptomatic bacteriuria strain Escherichia coli 83972, which fails to express type 1 fimbriae, due to a 4.25 kb fimB-fimD deletion, was reconstituted with a functional fim gene cluster and fimbrial expression was monitored through a gfp reporter. Each patient was inoculated with the fim+ or fim- variants on separate occasions, and the host response to type 1 fimbriae was quantified by intraindividual comparisons of the responses to the fim+ or fim- isogens, using cytokines and neutrophils as end-points. Type 1 fimbriae did not promote inflammation and adherence was poor, as examined on exfoliated cells in urine. This was unexpected, as type 1 fimbriae enhanced the inflammatory response to the same strain in the murine urinary tract and as P fimbrial expression by E. coli 83972 enhances adherence and inflammation in challenged patients. We conclude that type 1 fimbriae do not contribute to the mucosal inflammatory response in the human urinary tract.

Treatment of skin papillomas with topical alpha-lactalbumin-oleic acid.

BACKGROUND: We studied the effect on skin papillomas of topical application of a complex of alpha-lactalbumin and oleic acid (often referred to as human alpha-lactalbumin made lethal to tumor cells [HAMLET]) to establish proof of the principle that alpha-lactalbumin-oleic acid kills transformed cells but not healthy, differentiated cells. METHODS: Forty patients with cutaneous papillomas that were resistant to conventional treatment were enrolled in a randomized, placebo-controlled, double-blind study, in which alpha-lactalbumin-oleic acid or saline placebo was applied daily for three weeks and the change in the volume of each lesion was recorded. After this first phase of the study, 34 patients participated in the second phase, an open-label trial of a three-week course of alpha-lactalbumin-oleic acid. Approximately two years after the end of the open-label phase of the study, 38 of the original 40 patients were examined, and long-term follow-up data were obtained. RESULTS: In the first phase of the study, the lesion volume was reduced by 75 percent or more in all 20 patients in the alpha-lactalbumin-oleic acid group, and in 88 of 92 papillomas; in the placebo group, a similar effect was evident in only 3 of 20 patients (15 of 74 papillomas) (P<0.001). After the patients in the initial placebo group had been treated with alpha-lactalbumin-oleic acid in the second phase of the study, a median reduction of 82 percent in lesion volume was observed. At follow-up two years after the end of the second phase, all lesions had completely resolved in 83 percent of the patients treated with alpha-lactalbumin-oleic acid, and the time to resolution was shorter in the group originally assigned to receive alpha-lactalbumin-oleic acid than among patients originally in the placebo group (2.4 vs. 9.9 months; P<0.01). No adverse reactions were reported, and there was no difference in the outcomes of treatment between immunocompetent and immunosuppressed patients. CONCLUSIONS: Treatment with topical alpha-lactalbumin-oleic acid has a beneficial and lasting effect on skin papillomas.

PapG-dependent adherence breaks mucosal inertia and triggers the innate host response.

Mucosal pathogens differ from normal flora constituents in that they provoke a host response that upsets mucosal integrity. We investigated whether the elaboration of discrete adherence factors is sufficient to break the inertia of the mucosal barrier. PapG-mediated adherence was selected as an example, because P fimbrial expression characterizes uropathogenic Escherichia coli and because adherence starts the attack on the mucosal barrier. Patients were inoculated intravesically with transformed nonvirulent E. coli strains expressing functional P fimbriae (E. coli pap(+)) or mutant fimbriae lacking the adhesin (E. coli Delta papG). E. coli pap(+) was shown to activate the innate host response, and adherent gfp(+) bacteria were observed on excreted uroepithelial cells. E. coli Delta papG failed to trigger a response and was nonadhesive. We conclude that PapG-mediated adherence breaks mucosal inertia in the human urinary tract by triggering innate immunity and propose that this activation step differentiates asymptomatic carriage from infection.